Abstract Serotonergic neurons extensively innervate the visual system of the fruit fly Drosophila melanogaster, yet the role of serotonin signaling in visual processing is unknown. Identification of individual cells expressing serotonin receptors in visual system circuits will allow us to determine the contribution of serotonergic modulation to visual processing computations. Five genetically distinct serotonin receptors are expressed throughout the optic lobe and we identified specific neurons and visual processing pathways targeted by serotonin neuromodulation. Single-cell labeling mapped serotonin receptors to specific visual processing neurons: L2, L4, L5, T1, Lawf1, and Lawf2. Lamina monopolar cell 2 (L2) receives direct input from photoreceptors and expresses the serotonin receptor 5-HT2B. Calcium imaging confirmed an increase in baseline calcium in response to bath-applied serotonin in L2 and its electrically coupled partner, L1. L1 and L2 are the first-order neurons in the light-ON and light-OFF pathways, respectively, and both information streams feed into motion detection circuits. L1 neurons do not express serotonin receptors, demonstrating that indirect mechanisms such as gap junctions enable neuromodulators to influence cells in the absence of receptors. We tested whether serotonin modulates visual responses in L2 neurons and found that serotonin increased the magnitude of visually induced calcium transients. This suggests that neuromodulation may regulate salience to specific visual stimuli in the light-OFF pathway. In this work we tested acute effects of serotonin signaling and I propose to advance these findings by measuring physiological and transcriptomic changes in L2 neurons following chronic increases in serotonin signaling. Together, these data will allow us to compare serotonin modulation over multiple timescales. Additionally, these experiments will reveal molecular pathways and transcriptional programs downstream of serotonin signaling cascades that are important for long-term neuromodulation and may be relevant to mood elevation in humans. This work explores the molecular mechanisms of neuromodulation and will contribute to the understanding of how neuromodulatory signaling is integrated into sensory circuits.